The utility of the invention will be apparent from the following:
Plasminogen activators are enzymes which by their action upon plasminogen (a precursor of plasmin) result in the formation of plasmin. Plasmin in turn acts upon fibrin (or blood clots) to liquefy or dissolve the fibrin. Plasmin also causes lysis of fibrinogen which is a precursor of fibrin.
The foregoing effects play an important role in the natural fibrinolytic systems. They are also put to use in the therapeutic administration of plasminogen activators for the management, treatment or prophylaxis of thrombotic disease or other conditions where it is desirable to produce local fibrinolytic or proteolytic activity via the mechanism of plasminogen activation.
They may also find use in reagents for diagnostic, pathological or scientific tests involving fibrinolysis in vitro.
Two activators of human plasminogen are extensively used. The first of these is the bacterial enzyme streptokinase, which functions by a non-proteolytic mechanism. The second is the protease urokinase, which is mainly obtained from urine or cultured kidney cells and also by recombinant DNA procedures. These two compounds have the therapeutic disadvantage that their action is not confined to plasminogen associated with fibrin in blood clots. They act upon plasminogen generally in the circulation and, in consequence, produce widespread plasmin generation with extensive lysis of fibrinogen, the precursor of fibrin. This may in turn lead to a bleeding state as a result of ineffective normal coagulation of the blood. Streptokinase, being a foreign antigen, elicits an immune response in the patient. The resulting antibodies neutralize the action of streptokinase on plasminogen and hence diminish its therapeutic efficacy.
Much attention has therefore recently been given to a third plasminogen activator, referred to generally as "tissue plasminogen activator" hereinafter referred to as "tPA". This enzyme is found in most human tissues and is identical to or indistinguishable from an enzyme that it also a characteristic secretory product of human melanoma cells cultured in vitro. Although it catalyzes the proteolytic activation of plasminogen in much the same way as does urokinase, tPA differs from urokinase in a number of important respects. The two enzymes are chemically dissimilar. They have different molecular weights and each fails to react with antibodies to the other enzyme. The catalytic action of tPA is enhanced by fibrin, whereas that of urokinase is not. tPA has the important property that it binds to fibrin, whereas urokinase fails to do so. These facts were recorded in a recent article entitled "Purification and characterisation of the plasminogen activator secreted by human melanoma cells in culture" by Dingeman C. Rijken et al. (J. of Biological Chem., 256, (13); 7035-7041). That same article also describes the preparation of tPA from a cultured human melanoma cell line as well as from normal human tissue. The purification procedure consisted of successive chromatography on zinc chelate-agarose, concanavalin A-agarose and sephadex G-150 in the presence of detergent.
The tendency for tPA to bind to fibrin, its greatly enhanced fibrinolytic action in the presence of fibrin and, when compared to urokinase and streptokinase, its relatively inefficient function as a plasminogen activator in the absence of fibrin, combine to make tPA a plasminogen activator of choice for human thrombolytic therapy. Interactions between fibrin and tPA to a considerable extent localize the plasmin generation to the site of the clot and mitigate or avoid the consequences of promiscuous plasminogen activation as observed when urokinase or streptokinase is used. Furthermore, tPA is a protein which is immunochemically compatible with man, whereas streptokinase is not, being a foreign protein.
Recent reports, e.g., "Specific lysis of an iliofemoral thrombosis by administration of extrinsic (tissue type) plasminogen activator" by W. Weimar et al. (Lancet, November 7, 1981, page 1018), F. van der Werf et al. (New England J. of Med., (10); 609-613 (1984)) and the SPECIAL REPORT on the "Thrombolysis in Myocardial Infarction Trial" published in New England J. of Medicine, 312, (14); 932-936 (1985), testify to the clinical usefulness of tPA in the treatment of human thrombotic diseases.
According to P. Wallen et al, (Prog. Chem. Fibrinolysis Thrombolysis 5, 16-23 (1981)) tPA, occurs in two different forms, one being a single-chain form which is converted by plasmin or trypsin into two chains linked by disulphide bridges, and which those authors consider to be a degradation product of the former.
There are reasons why the single-chain form is rather to be considered the precursor of tPA. It will therefore be referred to herein as pro-tPA.
It has been found that the two molecular forms of tPA, in the presence of fibrin, exhibit very similar lysis times, because once the pro-tPA has been converted into tPA, it acts on the clot in the same manner as if it had been adsorbed in the form of tPA in the first instance. This observation is also in accordance with the work of other researches, e.g., Dingeman, Rijken, Hoylaerts and Collen (J.Biol.Chem., 257, 2920-2925 (1982), European patent application No. 0041 766).
The conversion of pro-tPA into tPA can be inhibited if desired (even in the presence of enzymes which would normally catalyze such conversion) by aprotinin, which is a protease inhibitor.
The therapeutic value of tPA as well as of pro-tPA is now generally accepted. However, until recently no method was known which would lend itself readily to the purification of two-chain tPA on a commercial scale, nor of the single chain form herein referred to as "pro-tPA".
Human tPA is present in tissue extracts, vessel perfusates and mammalian cell cultures. More recently, attention has been given to secure the expression of tPA by recombinant DNA technology in certain mammalian host cells, yeasts and bacteria. In all cases, the tPA enzyme is present in very low concentrations, usually of the order of a few milligrams per liter of culture fluid. Thus, to produce tPA efficiently on a commercial scale, a method is required to easily extract the tPA enzyme occurring in small quantities in a complex mixture of interfering proteins, cell debris and the like which are present in the cell culture medium. Moreover, such a method must achieve a high recovery of tPA from the harvest fluid and produce a product of high purity if the enzyme is to be administered intravenously. Also, a method must permit a high rate of production if it is to be used on a commercial scale.
A method proposed by Dingeman et al. (J.Biol. Chem., 256, (13), 7035-7041) uses a cumbersome three-step procedure giving a low rate of production because it incorporates a size exclusion chromatographic step. While this method produces a high degree of purification, the quoted recovery of tPA is relatively low.
The method employed by Wallen et al. referred to above (loc.cit,) produces slightly higher yields but much poorer purification. (Eur. J.Biochem., 132, 681-686 (1983).
Nielsen et al. (The EMBO J., 2, (1), 115-119) use immobilized monoclonal antibodies to tPA in a single step affinity purification. By that method improved yields and a reasonable purification are attained, compared with the above. However, the long term stability of monoclonal antibodies after repeated use for the immuno purification of tPA leaves much to be desired in that there is a danger of leakage of foreign proteins into the final tPA preparation.